A novel family of DNA mismatch-specific endonucleases from plants have been found (Oleykowski, et al. (1998) Nucl. Acid Res. 26:4597–4602; Yang, et al. (2000) Biochem. 39:3533–3541). Celery was found to be enriched in this endonuclease activity and the purified enzyme was accordingly named CEL I (Oleykowski, et al. (1998) supra; Yang, et al. (2000) supra). CEL I cleaves DNA at the 3′-side of sites of base-substitution mismatch and DNA distortion.
CEL I is useful in mismatch detection assays that rely on nicking and cleaving duplex DNA at insertion/deletion and base-substitution mismatches (Oleykowski, et al. (1998) supra; Yang, et al. (2000) supra; Kulinski, et al. (2000) BioTechniques 29:44–48; Colbert, et al. (20001) Plant Physiol. 126:480–484; Sokurenko, et al. (2001) Nucl. Acids Res. 29:e111; U.S. Pat. No. 5,869,245).
Purified preparations of CEL nuclease identified as CEL I contain two different protein species, CEL I and CEL II (Yang, et al. (2000) supra; U.S. Pat. No. 5,869,245). One species, called CEL I, has an apparent molecular weight of 43 kDa as determined by SDS-PAGE. Removal of N-linked oligosaccharides with Endo Hf reduces the molecular weight to 29 kDa. CEL I was partially sequenced and the gene encoding CEL I was isolated from a celery cDNA library, sequenced, and cloned into E. coli (Yang, et al. (2000) supra; U.S. Pat. No. 5,869,245). CEL II has an apparent molecular weight of 39 kDa as determined by SDS-PAGE and removal of N-linked oligosaccharides reduces the molecular weight to 37 kDa. Chromatographic separation of CEL I and CEL II has not been described and isolated preparations of CEL I nuclease contain varying ratios of CEL I and CEL II enzymes.
The present invention relates to the isolation and characterization of CEL I and CEL II from preparations of CEL I nuclease mixtures of celery. CEL I and CEL II enzymes are differentiated in a DNase solubilization assay; CEL I prefers acidic conditions whereas CEL II prefers alkaline reaction conditions.